Our ability to treat cancer, the second leading cause of death in the USA, remains woefully inadequate and, while novel therapies must be designed, optimization of existing therapies must be a complimentary priority. One underutilized class of chemotherapeutics is demethylating agents, currently the standard of care for several leukemias. An array of in vitro and animal model experiments have shown promise for these drugs in the treatment of solid tumors, however a limited understanding of their clinical efficacy has limited their use. Demethylating agents inhibit DNA methyltransferases, which methylate cytosines in CpG dinucleotides. CpG sites are concentrated in regions known as CpG islands, which are associated with the promoters of most human genes, are typically unmethylated, and serve as sites for transcriptional initiation. When CpG islands are methylated, the recruitment of RNA Polymerase II (Pol II) is inhibited, and the associated gene silenced. Cancer cells generally have aberrant DNA methylation profiles, including genomic hypomethylation and local hypermethylation of tumor-suppressor gene promoters. However, relief of promoter hypermethylation accounts for few transcriptional changes upon treatment with demethylating agents, and the largest portion of methylation changes in cancer occur not within CpG islands, but in flanking regions known as CpG island 'shores'. The role of DNA methylation in shores is not well established, but evidence suggests that shore methylation may regulate the release of Pol II from a paused to actively elongating state. After initiation, Pol II tracks 40-60bp before pausing, awaiting recruitment of various factors to advance to productive mRNA elongation. Studies have demonstrated a correlation between shore demethylation and enhanced transition of paused Pol II to active elongation, and in preliminary studies we have shown that Pol II pauses at shore/island transitions where methylation starkly increases. Pol II also tracks and pauses in the upstream antisense direction in a similarly regulated process referred to as divergent transcription that we hypothesize serves to prevent the spread of methylation into CpG islands. This proposal seeks to test the hypothesis that drug- induced demethylation of CpG island shores leads to the escape of RNA Polymerase II from a paused state, resulting in gene activation, and an increase in divergent transcription, both of which act to inhibit remethylation following drug withdrawal. We will test this model by treating MCF7 breast cancer cells with demethylating agents and monitoring methylation and transcriptional changes following treatment, both genome-wide, and at specific loci. We will then correlate these changes within various categories of genomic features to determine their relationship, e.g. if downstream shore hypomethylation corresponds to increased gene body transcription. These experiments will define how demethylation therapy changes transcription to inform design of novel chemotherapeutics and expand use of demethylating agents to solid tumors.